In the classification of a variety of fatty acids from a structural viewpoint, those having a long fatty acid chain comprising about 20 carbons in the molecule and containing two or more unsaturated sites (double bonds) are termed as polyunsaturated fatty acids (PUFAs). Alternatively, based on its extreme significance for the maintenance of human health from a nutritional viewpoint, some fatty acids are often expressed as essential fatty acids (EFAs). The definition of EFA signifies, in a narrow sense, linoleic acid (LA) and α-linolenic acid (ALA) that cannot be synthesized by humans and thus must be ingested through food, and in a broad sense, it also includes their metabolites, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Among them, a series of fatty acids produced with ALA as the parent fatty acid are termed as n3-series PUFAs, which include EPA and DHA.
On the other hand, as shown in FIG. 1, similar fatty acids produced with LA as the parent fatty acid are termed as n6-series PUFAs, in which, specifically, LA is metabolized by Δ6-desaturase to γ-linolenic acid (GLA), then by a carbon-chain elongase to DGLA, and further by Δ5-desaturase to AA.
The physiological role of these n6-series PUFAs have been extensively studied, and GLA, among them, has been demonstrated to be useful in skin disorders (Cosmetic & Toiletries, Nissen H P, 1995, 119: 71), diabetes mellitus and its complications (Diabetic Medicine, Jamal G A, 1990, 7: 319), rheumatoid arthritis (Arthritis Rhermatism, Zurier R B, 1996, 39: 1808) and the like. In connection with the molecular mechanism in the physiological function of GLA, it has been proposed, as shown in FIG. 1, that the active molecule is DGLA which is a carbon chain-elongated metabolite of GLA, the 1-series prostaglandin (PG1) which is a cyclooxygenase metabolite of DGLA, or 15-hydroxyeicosatrienoic acid (15-HETrE) which is a lipoxygenase metabolite of DGLA.
On the other hand, on the physiological activity of DGLA, part of the effect has been confirmed in vivo or in vitro: in vivo, effects of inhibiting platelet agglutination (British Medical Journal, Kernoff P B A, 1977, 2: 1441), delayed-type footpad edema (Lipids, Taki H., 1993, 28: 873), blood pressure increase (Lipids, Cedric H., 1984, 19: 699) and other effects have been reported, and in vitro, effects of inhibiting the production of cytokines such as interleukin-2, -10 and tumor necrosis factor (TNF-α) (Immunology, Maaike M B W D, 2003, 110: 348; The Journal of Immunology, Deniela S, 1989, 143: 1303), leukotriene production (Archives of Dermatological Research, Iversen L, 1992, 284: 222), and T cell growth (Prostaglandin Leukotrienes and Essential Fatty Acids, Zurier R B, 1999, 60: 371) and other effects have been reported.
The relationship of the physiological functions of living organisms (in particular, the skin) and PUFAs have been investigated in various fields. For example, it has been demonstrated that when animals such as rats are kept under an EFA-deficient condition for a long period of time, they may develop skin scale, decreased moisture, increased moisture loss and the like, and specifically abnormal skin functions. It is also suggested that PUFAs are deeply involved in various skin diseases such as eczema, contact dermatitis and UV-derived skin damages. Furthermore, GLA is useful in the prevention and treatment of various diseases, as described above, and among them, it has been proved, GLA is useful in skin diseases, specifically in atopic dermatitis (American Journal of Clinical Nutrition, Harrobin D F, 2000, 71: 367).
At first, it was found that in the serum of patients with atopic dermatitis, the amount of LA in the serum is increased as compared to normal healthy subjects, and conversely, at that time, the amounts of DGLA and AA that are metabolites from LA were found to be decreased. This fact strongly suggests the possibility that the function of Δ6-desaturase, an enzyme that converts LA to GLA, in the n6-series PUFA metabolic pathway is decreased in patients with atopic dermatitis.
Thus, it can be estimated that the ingestion of PUFAs which are located downstream to the Δ6-desaturase can improve diseases accompanied by abnormal PUFA metabolism, and it was investigated whether the ingestion of GLA, among them, which is relatively abundant in nature and abundant in the seeds of Oenothera, Ribes nigrum and Borrago officinalis can improve various conditions of atopic dermatitis.
As a result, it was reported in a human study that the oral ingestion of GLA (about 180-1440 mg/person/day) can improve the skin inflammation conditions or itching sensations of atopic dermatitis, and specifically it was more effective in patients who took a high dose of 720 or 1440 mg/person/day. The finding at the time confirmed that GLA can effectively increase the amounts of DGLA and AA in the living body, more effectively the amount of DGLA, and that there is a positive correlation between the improvement in disease conditions and DGLA (Prostaglandins Leukotrienes and Medicine, Mauku M S, 1982, 9: 615; The Lancet, Wright S, 1982, 20: 1120).
The effectiveness of GLA has also been confirmed in NC/Nga mice, an animal model of atopic dermatitis, and the oral ingestion of GLA (about 1250 mg/kg/day) has also been found to have effects of inhibiting the formation of atopic dermatitis and IgE production (Abstract of the 50th General Meeting of the Japanese Society of Allergology, Zui Hamada, 2000, pp. 999). As described above, the ingestion of GLA, among the n6-series PUFAs, is effective for the improvement of atopic dermatitis, and it has been estimated that preferably the ingestion of DGLA that is believed to be the active substance is more effective.
However, there remains a possibility that the appropriate establishment of an optimum dosage has not been made in GLA ingestion intended for the correction of abnormal PUFA metabolism such as atopic dermatitis mentioned above. In guinea pigs, a phenomenon was observed that the dosage and the amount increased of PUFAs in the living body does not correlate, i.e. in the administration of GLA at a highly excessive amount of about 3200 mg/kg/day, the amount of DGLA in the epidermis decreased as compared to that when about 400 mg/kg/day of GLA was ingested (Prostaglandins Leukotrienes and Essential Fatty Acids, Navarette R, 1992, 46: 139). This suggests a possibility that the ingestion of a large amount of GLA may inhibit the conversion of GLA to DGLA.
Thus, when a large amount of GLA is ingested, the amount of DGLA in the living body may tend to decrease rather than to increase, posing a risk of aggravating atopic dermatitis. Furthermore, it is known that there exists an individual difference in the metabolism of GLA in humans. When GLA was administered to human patients with atopic dermatitis, the degree of enhancement in the amount of DGLA in the erythrocyte membrane varied with individuals, and in the patient group having enhancement in the amount of DGLA the dermatitis condition improved, whereas no improvement was noted in the patient group having no enhancement in the amount of DGLA (British Journal of Dermatology, Henz B M, 1999, 140: 685). This means that the ingestion of GLA is not necessarily effective for increasing the amount of DGLA, and that in the treatment of atopic dermatitis, the optimum amount of GLA may not be the optimum amount of DGLA.
On the other hand, it has been confirmed that the ingestion of DGLA leads to increases in the amount of DGLA in the living body in a dose dependent manner (Abstract of the 58th Meeting of the Japanese Society of Nutrition and Food Science, Chika Horikawa, 2004, pp. 219), and thus even if DGLA was ingested in a large amount, it is hard to conceive that the amount of DGLA in a living body decreases. Furthermore, in the inhibition of metabolic enzymes by GLA described above, originally the activity of the enzyme is considered to be relatively high, which indicates a possibility that carbon-chain elongation enzymes possibly free of activity reduction due to aging may be affected by the substrate environment or other factors as long as they participate in enzyme reactions.
As a result, it is likely that the amount of DGLA when GLA was ingested may vary depending on various conditions, which strongly suggests the difficulty of appropriately controlling the treatment of atopic dermatitis by the ingestion of GLA. Thus, from the viewpoint of safety and effectiveness, the direct ingestion of DGLA per se is more preferred than the ingestion of GLA in the treatment or prevention of atopic dermatitis, in which it is further considered to be very important to provide an optimum dosage.
However, some meat, eggs and seafoods contain DGLA, but they are limited in the number of types, and for vegetarians, ingestion of DGLA from natural products is very difficult. Furthermore, DGLA occurs in nature, but the amount is very limited and mass production is difficult, and thus it is very difficult to demonstrate its effect on atopic dermatitis using DGLA as in the GLA study mentioned above. However, as far as we know, there is no direct demonstration that atopic dermatitis is improved by DGLA per se, and though DGLA has been demonstrated to have various physiological effects in in vivo tests or in vitro tests using various animal- or human-derived cell lines, as described above, none of the tests are considered to simulate atopic dermatitis, and thus there is no definite answer to whether DGLA can improve atopic dermatitis or not.
As shown in Japanese Patent No. 3354581, by inventing a method of obtaining microorganisms deficient in ™5-desaturase and producing DGLA lipids by fermentation of the microorganisms, the present inventors have enabled mass production of a triglyceride SUNTGD of which about 40% of constituent fatty acids comprises DGLA, and thus have overcome the previous difficulties in obtaining the raw material of DGLA.
In recent years, it has been demonstrated that eosinophil infiltration occurs at inflammatory regions in various diseases including skin diseases such as atopic dermatitis, eczema and psoriasis, respiratory system diseases such as bronchial asthma, chronic obstructive pulmonary disease (COPD), hypersensitivity pneumonitis and eosinophilic pneumonitis, and digestive system diseases such as eosinophilic gastroenteritis and ulcerative colitis, and thus eosinophils have been implicated to be deeply involved in the formation and progression of these disease conditions. Under these circumstances, attempts have been made to prevent and/or treat these diseases by inhibiting the steps of eosinophil infiltration into the tissues of the lesion, specifically eosinophil activation, adhesion to the endothelium, extravascular migration, and movement of chemotactic factors into the lesion (American Journal of Clinical Dermatology, Chari S, 2001, 2: 1; Paediatric Respiratory Reviews, McMillan R M, 2001, 2: 238; Agents Actions, Rask-Madsen J, 1992, C37; Japanese Unexamined Patent Publication (Kokai) No. 8-3036).
For example, it has been found that steroid external preparations and immunosuppressive external preparations for which clinical usefulness has been confirmed for the treatment of skin diseases can alleviate the aggravation of skin conditions in an atopic dermatitis animal model, NC/Nga mice, and also to suppress significantly the number of eosinophils infiltrating into the skin lesions (Japanese Journal of Pharmacology, Hiroi J, 1998, 76: 175). Also, as a substance that is a chemotactic factor having a property of accumulating eosinophils into the lesions, leukotriene B4 can be mentioned, and this substance has been found to be synthesized by 5-lipoxygenase.
In contrast, 5-lipoxygenase inhibitors suppress the production of leukotriene B4 by inhibiting the activity of this enzyme, and also suppress the subsequent biological event of eosinophil infiltration. As a result, it has been found, the compounds can alleviate respiratory disorders of nocturnal asthma in which leukotrienes and eosinophils are deeply involved, indicating their usefulness in respiratory system diseases (American Journal of Respiratory and Critical Care Medicine, Wenzel S E, 1995, 152: 897). Thus, these pharmaceutical drugs have been demonstrated to be useful in the prevention and/or treatment of various diseases by controlling abnormal eosinophil conditions, whereas there are problems with safety and the methods for using them.
There are side effects in using steroid external preparations, such as blushing and atrophy of the skin during use and the rebound phenomenon in which the suspension of administration may aggravate dermatitis, whereas immunosuppressive external preparations may facilitate skin tumors and their efficacy is greatly affected by the application site and the state of the barrier function of the skin (Journal of the Japanese Dermatological Association, Masutaka Furue, 2004, 114: 135), and 5-lipoxygenase inhibitors must be orally administered as many as four times a day and their ingestion over a long period of time may cause dyspepsia (ZYFLO™ FLIMTAB™, package insert, Abbott Laboratories, 1998).
Under these circumstances, materials have long been sought that are medically useful, which can be used safely by everyone, and can effectively inhibit the function of eosinophils.
Considering medically or nutritionally useful food ingredients, there can be mentioned eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) that are n3-series polyunsaturated fatty acids as shown in FIG. 2 as one of the candidate materials. These ingredients are relatively abundant in marine animal oils, specifically fish oils such as bonito and sardine oils, which have been ingested by mankind for a long period of time and are naturally very safe. Also, extensive studies have been made on the physiological functions thereof, and such usefulness has been found as the effect of inhibiting blood clots for the former and as the effect of enhancing the learning function has been demonstrated for the latter (New Developments in Functional Lipids (Kinousei Shishitsuno Shintennkai), Osamu Suzuki, 2001).
As another useful physiological effect, there can be mentioned an anti-allergy effect (The European Respiratory Journal, Nagatsuka T. 2000, 16: 861; The Journal of Infectious Diseases, McMurray D N, 2000, 182: 861), and one of the mechanisms thereof is proposed to be an effect on the function of immunocompetent cells. As one of the studies that support this, as shown in Japanese Unexamined Patent Publication (Kokai) No. 10-1434 to Yazawa et al., the effect of EPA and DHA on eosinophil migration has been investigated, and revealed that the intraperitoneal administration of 100 mg of EPA ethylester or 50 mg of DHA ethylester can inhibit eosinophil migration in the delayed type allergy in guinea pigs.
However, this experiment is a confirmation of a biological reaction when EPA and DHA were intraperitoneally administered, and cannot be considered to be an experiment that simulates a situation in which they were actually ingested as a foodstuff. When intraperitoneal administration and oral administration are compared, it can be easily conjectured that the concentrations of EPA and DHA are overwhelming higher in the abdominal cavity, in which eosinophils can infiltrate, in the former, i.e. the physiological activity can be more easily detected. Thus, it is unknown whether oral ingestion provides such effects or not.
There are also problems of parts being easily oxidized and offensive odors resulting from the structural characteristics of EPA and DHA. EPA and DHA have unsaturated bonds in their molecules, or parts that are easily oxidized: five in EPA and six in DHA. By undergoing oxidation, there is quality deterioration, and the possibility cannot be ruled out that not only does it impair the physiological functions described above, but the oxidation products formed may be detrimental to a living body. Furthermore, these oxidation products of PUFAs are known to emit exceptionally bad orders, and become worse with accelerated oxidation over time.
As a countermeasure against easily oxidized EPA and DHA, antioxidants, deodorants, masking agents, etc., have been contrived. However, because of various problems in that their efficacy for preventing oxidation and that its duration are not satisfactory and some additives may cloud the appearance, none are considered effective preventive measures (Japanese Unexamined Patent Publication (Kokai) No. 2-55785, Japanese Unexamined Patent Publication (Kokai) No. 3-100093, Japanese Unexamined Patent Publication (Kokai) No. 2004-137420). Despite the useful biological activity of EPA and DHA, it is difficult to stably maintain their quality even with a variety of measures, which represents one of the reasons that limit the range of application into foodstuffs.
From the foregoing, food materials that are medically useful and that are excellent in safety and quality stability are being sought after.
In recent years, because of changes in the environment and eating habits, genetic factors and the like, the number of patients afflicted with allergic diseases has increased. Pathological conditions of allergic diseases are roughly divided into type I to type IV based on the mechanism of pathogenesis and the related functional molecules. Mast cells are considered to be closely related to type a I allergy, also termed as delayed type allergy, among them. In a type I allergy, when a living body is exposed to a certain antigen, an antigen-specific IgE antibody is produced by B cells via antigen presenting cells and helper T cells.
Subsequently, IgE antibody binds to the surface of mast cells to enter into the state of a guard against the reentry of antigen. Mast cells in this state capture antigen that enter the surface of the membrane, and initiate degranulation so as to release various chemical mediators such as histamine and leukotrienes. The subsequent binding of these chemical mediators to the receptors causes so-called “allergic reactions” i.e. physiological phenomena that are detrimental to humans such as edema, reddening, itching, airway resistance and enhanced mucous secretion.
Under these circumstances, various attempts for alleviating allergic reactions have been made, by inhibiting the function of mast cells, specifically by a method of suppressing the degranulation of mast cells to suppress the release of chemical mediators, a method of suppressing the synthesis of chemical mediators, or a method of inhibiting the binding of the released chemical mediators and receptors, specifically by suppressing the actions of chemical mediators produced by mast cells. It is recognized now that many of the pharmaceutical agents used for alleviating itching associated with atopic dermatitis, and treating pollinosis, allergic conjunctivitis etc., are based on any of the mechanisms described above (Internet HP “The Rheumatism & Allergy Information Center”, Maki Hasegawa, 2005.04.04, Allergic inflammatory diseases—New approaches, Motohiro Kurosawa, 1994).
However, some reports describe that not only the function of the above chemical mediators produced by mast cells, but the number per se of mast cells identified in the lesion are changed. For example, it has been found that the differentiation and proliferation of mast cells in the nasal mucosa of patients with allergic rhinitis are more frequent than those with nonallergic diseases (Journal of the Oto-Rhino-Laryngological Society of Japan (Nippon Jibiinkoka Gakkai Kaiho), Naoshi Yoshida, 2001, 104: 504). It has also been revealed that patients who have asthma, the number of mast cells localized in bronchial smooth muscles are significantly greater than normal healthy subjects (Current Opinion in Allergy and Clinical Immunology, Peter B, 2003, 3: 45).
There are similar reports on skin diseases, for example increases in mast cells have been reported in lesions, etc., of basal cell carcinoma, psoriasis vulgaris and atopic dermatitis (Allergy, Shoso Yamamoto, 2000, 49: 455). In other words, as a means for suppressing and/or alleviating diseases closely related to enhanced mast cell count, it is important not only to control the amount and function of chemical mediators produced by inflammatory cells, specifically mast cells, but also to suppress the abnormal growth of the mast cells per se to keep the number of these cells at normal levels, and it is believed that by controlling the processes, more effective and multi-faceted prevention and treatment of diseases can be attained.
For example, it has been found that steroid external preparations, immunosuppressive external preparations or the like of which clinical significance has been recognized against allergic dermatitis, etc., not only exhibit significant improvement in the dermatitis symptom score associated with dermatitis, but also significantly suppress increases in the number of mast cells in the lesions of dermatitis in the NC/Nga mice which is an animal model of allergic dermatitis (Japanese Journal of Pharmacology, Hiroi J, 1998, 76: 175). This also strongly suggests that the therapeutic effect and mast cell count are correlated, and supports the importance of regulating mast cell count at an appropriate level.
Thus, these pharmaceutical drugs have been demonstrated to be useful in the prevention and/or treatment of various mast cell-related diseases such as allergic diseases, whereas they have problems in safety and methods of use. Steroid external preparations have side effects, such as blushing, atrophy of the skin, and a rebound phenomenon in which suspending administration may aggravate dermatitis, whereas immunosuppressive external preparations have problems in that their use may facilitate skin tumors and their efficacy is greatly affected by the application site and the state of the barrier function of the skin (Journal of the Japanese Dermatological Association, Masutaka Furue, 2004, 114: 135).
Under these circumstances, materials have long been sought that are medically useful, that can be used safely by everyone, and that can effectively suppress enhancement in the number of mast cells.
Patent document 1: Japanese Patent No. 3354581
Patent document 2: Japanese Unexamined Patent Publication (Kokai) No. 8-3036
Patent document 3: Japanese Unexamined Patent Publication (Kokai) No. 2-55785
Patent document 4: Japanese Unexamined Patent Publication (Kokai) No. 3-100093
Patent document 5: Japanese Unexamined Patent Publication (Kokai) No. 2004-137420
Nonpatent document 1: Cosmetic & Toiletries, Nissen H P, 1995, 119: 71
Nonpatent document 2: Diabetic Medicine, Jamal G A, 1990, 7: 319
Nonpatent document 3: Arthritis Rhermatism, Zurier R B, 1996, 39: 1808
Nonpatent document 4: British Medical Journal, Kernoff P B A, 1977, 2: 1441
Nonpatent document 5: Lipids, Taki H., 1993, 28: 873
Nonpatent document 6: Lipids, Cedric H., 1984, 19: 699
Nonpatent document 7: Immunology, Maaike M B WD, 2003, 110: 348
Nonpatent document 8: The Journal of Immunology, Deniela S, 1989, 143: 1303
Nonpatent document 9: Archives of Dermatological Research, Iversen L, 1992, 284: 222
Nonpatent document 10: Prostaglandin Leukotrienes and Essential Fatty Acids, Zurier R B, 1999, 60: 371
Nonpatent document 11: American Journal of Clinical Nutrition, Harrobin D F, 2000, 71: 367
Nonpatent document 12: Prostaglandins Leukotrienes and Medicine, Mauku M S, 1982, 9: 615
Nonpatent document 13: The Lancet, Wright S, 1982, 20: 1120
Nonpatent document 14: Abstract of the 50th General Meeting of the Japanese Society of Allergology, Zui Hamada, 2000, pp. 999
Nonpatent document 15: Prostaglandins Leukotrienes and Essential Fatty Acids, Navarette R, 1992, 46: 139
Nonpatent document 16: British Journal of Dermatology, Henz B M, 1999, 140: 685
Nonpatent document 17: Abstract of the 58th meeting of the Japanese Society of Nutrition and Food Science, Chika Horikawa, 2004, pp. 219
Nonpatent document 18: New Developments in Functional Lipids (Kinosei Shishitsuno Shintennkai), Osamu Suzuki, 2001
Nonpatent document 19: γ-Linolenic Acid, Recent Advances in Biotechnology and Clinical Applications, Hundy Y S, 2001
Nonpatent document 20: American Journal of Clinical Dermatology, Chari S, 2001, 2: 1
Nonpatent document 21: Paediatric Respiratory Reviews, McMillan R M, 2001, 2: 238
Nonpatent document 22: Agents Actions, Rask-Madsen J, 1992, C37 (Japanese Unexamined Patent Publication (Kokai) No. 8-3036)
Nonpatent document 23: Japanese Journal of Pharmacology, Hiroi J, 1998, 76: 175
Nonpatent document 24: American Journal of Respiratory and Critical Care Medicine, Wenzel S E, 1995, 152: 897
Nonpatent document 25: Journal of the Japanese Dermatological Association, Masutaka Furue, 2004, 114: 135
Nonpatent document 26: ZYFLO™ FLIMTAB, the package insert, Abbott Laboratories, 1998
Nonpatent document 27: The European Respiratory Journal, Nagatsuka T. 2000, 16; 861
Nonpatent document 28: The Journal of Infectious Diseases, McMurray D N, 2000, 182: 861
Nonpatent document 29: British Medical Journal, Kernoff P B A, 1977, 2: 1441
Nonpatent document 30: The Journal of Immunology, Deniela S, 1989, 143: 1303
Nonpatent document 31: Archives of Dermatological Research, Iverson L, 1992, 284: 222
Nonpatent document 32: Maki Hasegawa, 2005.04.04, Allergic inflammatory diseases—New approaches, Motohiro Kurosawa, 199
Nonpatent document 33: Journal of the Oto-Rhino-Laryngological Society of Japan (Nippon Jibiinkoka Gakkai Kaiho), Naoshi Yoshida, 2001, 104: 504
Nonpatent document 34: Current Opinion in Allergy and Clinical Immunology, Peter B, 2003, 3: 45
Nonpatent document 35: Prostaglandins Leukotrienes and Essential Fatty Acids, Gueck T. 2003, 68: 317
Nonpatent document 36: Veterinary Dermatology, Gueck T. 2004, 15: 309
Nonpatent document 37: Allergy, Shoso Yamamoto, 2004, 49: 45.